This invention relates generally to methods for fabricating covalently bonded hydrophilic coatings on hydrophobic surfaces.
Polystyrene divinylbenzene (PS-DVB) based chromatography materials, such as POROS (Registered) materials, manufactured by PerSeptive Biosystems (Cambridge, Mass.), have been demonstrated to be useful in the separation of macromolecules. U.S. Pat. No. 5,019,270 (1991). However, the inherent hydrophobic character of hydrocarbon-based chromatographic supports, such as PS-DVB, is a serious impediment to their use in protein separations. Boardman and Patridge, Biochem J., 59:543 (1955). Hydrophobic interactions between the support and proteins are sufficiently strong that proteins may be denatured either upon adsorption or during elution. Additionally, PS-DVB chromatography supports are difficult to derivatize, since they have no active functional groups for direct derivatization. Frechet et al., "Future Trends in Polymer Science and Technology", E Martuscelli, Ed., Technomic Publishing Co., Inc., Basel, 1987.
The aromatic rings of the monomers have been derivatized, before polymerization, to produce a variety of functionalized PS-DVB packing materials, which were not useful in protein separations. Frechet et al., Pure & Appl. Chemo, 60:353 (1988). Functional groups attached to the aromatic rings cannot mask the surface of PS-DVB, and the hydrophobic residues of proteins still can interact with the hydrophobic surface. PS-DVB beads have been modified by attaching hydrophilic functionalities on both the aromatic ring and the polymer backbones. Yang et al., Chromatographia, 20:735 (1985); Yang and Verzele, J. Chromatogr., 387:197 (1987); and Yang and Verzele, J. Chromatogr., 391:383 (1987). Using these modified PS-DVB materials, however, organic solvents had to be added to the mobile phase during chromatography because of hydrophobic interactions.
Sulfonation of PS-DVB, followed by coating with polyethyleneimine, after crosslinking and quaternization, produced a strong anion-exchange packing material. Rounds et al., J. Chromatogr., 397:25 (1987). The disadvantage of this coating is that the polyamine polymer could only be used to make anion-exchangers. It was impossible to prepare a broad spectrum of PS-DVB based packings using this coating.
Pharmacia (Uppsala, Sweden) has prepared functionalized PS-DVB based packings for protein separation under the trade name MONOBEADS by proprietary procedures. A grafted copolymer of polyoxyethylene and crosslinked polymers was constructed as a support to bind proteins in some applications, but very few chromatographic properties have been reported. E. Bayer, German Pat. No. DE 3,500,180 (1986); and U.S. Pat. No. 4,908,405 (1990).
Molecules of a solute comprising hydrophilic and hydrophobic domains were adsorbed onto a hydrophobic polymeric support, such as PS-DVB, and crosslinked to produce a hydrophilic coating on the support. U.S. Pat. No. 5,030,352 (1991). The crosslinking and polymerization of epichlorohydrin and glycidol was reported. Additionally, the formation of a crosslinked coating of glycidyl methacrylate adsorbed onto PS-DVB followed by derivatization of the coating with methacrylic acid in the presence of ammonium persulfate and tetramethylethylene diamine was reported. The reference discloses that the molecules of the coating are chemically crosslinked among each other to form a continuous layer, and the crosslinked layer is adsorbed to the PS-DVB surface by hydrophobic-hydrophobic interaction. The reference failed to suggest that covalent bonds were formed between the coating and PS-DVB.
It is an object of the invention to provide methods for forming covalent bonds between hydrophilic coatings and the surface of hydrophobic materials, such as PS-DVB. It is another object of the invention to provide covalent hydrophilic coatings on hydrophobic surfaces, such as PS-DVB polymers, which are sufficiently hydrophilic to mask the hydrophobic surfaces, and which permit the polymer to be used in protein separation without causing protein denaturation. It is a further object of the invention to provide covalent coatings which may be derivatized readily to produce a wide range of different functional groups for use in different chromatography applications.